Titania composite microspheres endowed with a size-exclusive effect toward the highly specific revelation of phosphopeptidome.

The efficient isolation of low-abundance phosphopeptides from complicated biological samples containing a significant quantity of nonphosphopeptides and proteins is essential for phosphopeptidome research but remains a great challenge. In this Article, magnetic composite microspheres comprising a magnetic colloidal nanocrystal cluster core and a mesoporous titania shell with an average pore diameter of 3.4 nm were modified by directly coating an amorphous titania shell onto the magnetite core, followed by converting the amorphous titania shell into a crystalline structure via a hydrothermal process at 80 °C. The as-prepared magnetic mesoporous titania microspheres possess a remarkable specific surface area that is as high as 603.5 m2/g, which is an appropriate pore size with a narrow size distribution and a high magnetic responsiveness. These outstanding features imply that the composite microspheres exhibit extraordinary performance in phosphopeptidome research, including high specificity toward phosphopeptides, an excellent size-exclusion effect against phosphoproteins, exceptional enrichment capacity, and efficient separation from mixtures. Encouraged by the experimental results, we employed this method to investigate the phosphopeptidome of snake venom for the first time. A total of 35 phosphopeptides was identified from the snake venom from the family Viperidae, accounting for 75% of the total identified peptides. This result represents the largest data set of the phosphopeptidome in snake venom from the family Viperidae.